Division of Gastroenterology, Zhongshan Hospital Affiliated to Xiamen University, Xiamen 361004, Fujian Province, China.
World J Gastroenterol. 2010 Mar 7;16(9):1070-5. doi: 10.3748/wjg.v16.i9.1070.
To investigate the regulation of mindin expression and the signaling pathway involved during inflammation.
C57BL/6 mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 6 d to induce acute colitis, and then the colon was harvested for histological analysis or for RNA isolation. mRNA expression of mindin and nuclear factor (NF)-kappaB p65 was analyzed by quantitative real time polymerase chain reaction (RT-PCR) and mindin expression construct was confirmed by Western blotting. Mouse macrophage and intestinal epithelial lineage cells were stimulated with different cytokines and toll-like receptor (TLR) ligands, before pNF-kappaB-luciferase activity was assessed using the Dual-Luciferase reporter assay system.
mRNA expression of mindin was upregulated 4.7 + or - 1.1 fold compared with the baseline during DSS-induced intestinal inflammation in the mice. Stimulation with CpG-ODN (a known TLR-9 ligand) induced 4.2 + or - 0.3 fold upregulation of mindin expression in RAW 264.7 cells. Full-length of mindin was cloned from cDNA of mouse mesenteric lymph node, then the pCMV-Mindin-Flag expression vector was established and the protein expression level was confirmed. Transfection of the mindin construct and stimulation with CpG-ODN significantly increased the NF-kappaB-luciferase activity by 2.5 + or - 0.3 and 4.5 + or - 0.5 fold in RAW264.7 and CMT93 cells, respectively (P < 0.01).
Mindin expression is upregulated during intestinal inflammation and may induce NF-kappaB promoter activation in a TLR-9 mediated manner.
研究 Mindin 表达的调节及其在炎症过程中涉及的信号通路。
用 3%葡聚糖硫酸钠（DSS）饮用水处理 C57BL/6 小鼠 6d 诱导急性结肠炎，然后采集结肠进行组织学分析或 RNA 分离。通过实时定量聚合酶链反应（RT-PCR）分析 Mindin 和核因子（NF）-κB p65 的 mRNA 表达，通过 Western blot 确认 Mindin 表达构建体。用不同细胞因子和 Toll 样受体（TLR）配体刺激小鼠巨噬细胞和肠上皮细胞系，然后使用双荧光素酶报告基因检测系统评估 pNF-κB-荧光素酶活性。
与小鼠 DSS 诱导的肠道炎症基础水平相比，Mindin 的 mRNA 表达上调了 4.7 ± 1.1 倍。CpG-ODN（已知的 TLR-9 配体）刺激诱导 RAW 264.7 细胞中 Mindin 表达上调 4.2 ± 0.3 倍。从鼠肠系膜淋巴结 cDNA 中克隆全长 Mindin，然后建立 pCMV-Mindin-Flag 表达载体并确认蛋白表达水平。Mindin 构建体的转染和 CpG-ODN 刺激分别使 RAW264.7 和 CMT93 细胞中的 NF-κB 荧光素酶活性增加 2.5 ± 0.3 和 4.5 ± 0.5 倍（P < 0.01）。
在肠道炎症期间，Mindin 表达上调，并且可能以 TLR-9 介导的方式诱导 NF-κB 启动子激活。
